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Seed germination is regulated by environmental factors, particularly water availability. Water deficits at the time of sowing impair the establishment of crop plants. Transcriptome and proteome profiling was used to document the responses of sunflower (Helianthus annuus) seeds to moderate water stress during germination in two hybrids that are nominally classed as drought sensitive and drought tolerant. Differences in the water stress-dependent accumulation reactive oxygen species and antioxidant enzymes activities were observed between the hybrids. A pathway-based analysis of the hybrid transcriptomes demonstrated that the water stress-dependent responses of seed metabolism were similar to those of the plant, with a decreased abundance of transcripts encoding proteins associated with metabolism and cell expansion. Moreover, germination under water stress conditions was associated with increased levels of transcripts encoding heat shock proteins. Exposure of germinating seeds to water stress specifically affected the abundance of a small number of proteins, including heat shock proteins. Taken together, these data not only identify factors that are likely to play a key role in drought tolerance during seed germination, but they also demonstrate the importance of the female parent in the transmission of water stress tolerance., (© 2020 John Wiley & Sons Ltd.)

Regulation of seed germination by dormancy relies on a complex network of transcriptional and post-transcriptional modifications during seed imbibition that controls seed adaptive responses to environmental cues. High-throughput technologies have brought significant progress in the understanding of this phenomenon and have led to identify major regulators of seed germination, mostly by studying the behaviour of highly differentially expressed genes. However, the actual models of transcriptome analysis cannot catch additive effects of small variations of gene expression in individual signalling or metabolic pathways, which are also likely to control germination. Therefore, the comprehension of the molecular mechanism regulating germination is still incomplete and to gain knowledge about this process we have developed a pathway-based analysis of transcriptomic Arabidopsis datasets, to identify regulatory actors of seed germination. The method allowed quantifying the level of deregulation of a wide range of pathways in dormant versus non-dormant seeds. Clustering pathway deregulation scores of germinating and dormant seed samples permitted the identification of mechanisms involved in seed germination such as RNA transport or vitamin B6 metabolism, for example. Using this method, which was validated by metabolomics analysis, we also demonstrated that Col and Cvi seeds follow different metabolic routes for completing germination, demonstrating the genetic plasticity of this process. We finally provided an extensive basis of analysed transcriptomic datasets that will allow further identification of mechanisms controlling seed germination., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

Temperature is the primary factor that affects seed dormancy and germination. However, the molecular mechanism that underlies its effect on dormancy alleviation remained largely unknown. In this study, we investigate hormone involvement in temperature induced germination as compared to that caused by after-ripening. Dormant (D) sunflower seeds cannot germinate at 10 °C but fully germinate at 20 °C. After-ripened seeds become non-dormant (ND), i.e. able to germinate at 10 °C. Pharmacological experiments showed the importance of abscisic acid (ABA), gibberellins (GAs) and ethylene in temperature- and after-ripening-induced germination of sunflower seeds. Hormone quantification showed that after-ripening is mediated by a decline in both ABA content and sensitivity while ABA content is increased in D seeds treated at 10 or 20 °C, suggesting that ABA decrease is not a prerequisite for temperature induced dormancy alleviation. GAs and ethylene contents were in accordance with germination potential of the three conditions (GA 1 was higher in D 20 °C and ND 10 °C than in D 10 °C). Transcripts analysis showed that the major change concerns ABA and GAs metabolism genes, while ABA signalling gene expression was significantly unchanged. Moreover, another level of hormonal regulation at the subcellular localization has been revealed by immunocytolocalization study. Indeed, ABA, protein Abscisic acid-Insensitive 5 (ABI5), involved in ABA-regulated gene expression and DELLA protein RGL2, a repressor of the gibberellins signalling pathway, localized mainly in the nucleus in non-germinating seeds while they localized in the cytosol in germinating seeds. Furthermore, ACC-oxidase (ACO) protein, the key ethylene biosynthesis enzyme, was detected in the meristem only in germinating seeds. Our results reveal the importance of hormone actors trafficking in the cell and their regulation in specialized tissue such as the meristem in dormancy alleviation and germination.

Dormant Arabidopsis ( Arabidopsis thaliana ) seeds do not germinate easily at temperatures higher than 10⁻15 °C. Using mutants affected in ethylene signaling ( etr1 , ein2 and ein4 ) and in the N-end-rule pathway of the proteolysis ( prt6 and ate1-ate2 ) we have investigated the effects of cold and ethylene on dormancy alleviation. Ethylene (10⁻100 ppm) and 2⁻4 days chilling (4 °C) strongly stimulate the germination of wild type (Col-0) seeds at 25 °C. Two to four days of chilling promote the germination at 25 °C of all the mutants suggesting that release of dormancy by cold did not require ethylene and did not require the N-end-rule pathway. One mutant ( etr1 ) that did not respond to ethylene did not respond to GA₃ either. Mutants affected in the N-end rule ( prt6 and ate1-ate2 ) did not respond to ethylene indicating that also this pathway is required for dormancy alleviation by ethylene; they germinated after chilling and in the presence of GA₃. Cold can activate the ethylene signaling pathway since it induced an accumulation of ETR1 , EINI4, and EIN2 transcripts, the expression of which was not affected by ethylene and GA₃. Both cold followed by 10 h at 25 °C and ethylene downregulated the expression of PRT6 , ATE1, ATE2, and of ABI5 involved in ABA signaling as compared to dormant seeds incubated at 25 °C. In opposite, the expression of RGA , GAI, and RGL2 encoding three DELLAs was induced at 4 °C but downregulated in the presence of ethylene.

Dormancy is an adaptive trait that blocks seed germination until the environmental conditions become favorable for subsequent vegetative plant growth. Seed dormancy is defined as the inability to germinate in favorable conditions. Dormancy is alleviated during after-ripening, a dry storage period, during which dormant (D) seeds unable to germinate become non-dormant (ND), able to germinate in a wide range of environmental conditions. The treatment of dormant seeds with ethylene (D/ET) promotes seed germination, and abscisic acid (ABA) treatment reduces non-dormant (ND/ABA) seed germination in sunflowers ( Helianthus annuus ). Metabolomic and transcriptomic studies have been performed during imbibition to compare germinating seeds (ND and D/ET) and low-germinating seeds (D and ND/ABA). A PCA analysis of the metabolites content showed that imbibition did not trigger a significant change during the first hours (3 and 15 h). The metabolic changes associated with germination capacity occurred at 24 h and were related to hexoses, as their content was higher in ND and D/ET and was reduced by ABA treatment. At the transcriptional level, a large number of genes were altered oppositely in germinating, compared to the low-germinating seeds. The metabolomic and transcriptomic results were integrated in the interpretation of the processes involved in germination. Our results show that ethylene treatment triggers molecular changes comparable to that of after-ripening treatment, concerning sugar metabolism and ABA signaling inhibition.

Temperature is an important environmental factor affecting seed dormancy and germination. The mechanism by which temperature induces germination in dormant seeds is however still unclear. Proteomic study has been performed in dormant sunflower seeds during imbibition at permissive and non-permissive temperatures for germination, 20 and 10 °C, respectively. Proteome analysis showed an increase of proteins belonging to metabolism and energy from the first hours of imbibition followed by a decrease of proteins involved in protein metabolism and seed storage in germinating compared to non-germinating seeds. Proteomic study was completed by polysome and proteasome activity assessment and enzymatic profiling on several altered proteins involved in metabolism and energy. Results showed that 20 °C treatment induced the activation of both protein synthesis and degradation processes, the latter being related to proteasome activity during the germination sensu stricto, and to other degradation processes such as proteases during the post-germination. Interestingly, enzymatic profiles showed that TCA cycle and glycolysis were more active in non-germinating seeds in the phase I of the germination sensu stricto. This result suggests the regulation of central metabolism activity in germinating seeds. The control of energy production during imbibition seems to be involved in molecular networks controlling seed dormancy and germination., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Sunflower (Helianthus annuus L.) seed dormancy is regulated by reactive oxygen species (ROS) and can be alleviated by incubating dormant embryos in the presence of methylviologen (MV), a ROS-generating compound. Ethylene alleviates sunflower seed dormancy whereas abscisic acid (ABA) represses germination. The purposes of this study were to identify the molecular basis of ROS effect on seed germination and to investigate their possible relationship with hormone signalling pathways. Ethylene treatment provoked ROS generation in embryonic axis whereas ABA had no effect on their production. The beneficial effect of ethylene on germination was lowered in the presence of antioxidant compounds, and MV suppressed the inhibitory effect of ABA. MV treatment did not alter significantly ethylene nor ABA production during seed imbibition. Microarray analysis showed that MV treatment triggered differential expression of 120 probe sets (59 more abundant and 61 less abundant genes), and most of the identified transcripts were related to cell signalling components. Many transcripts less represented in MV-treated seeds were involved in ABA signalling, thus suggesting an interaction between ROS and ABA signalling pathways at the transcriptional level. Altogether, these results shed new light on the crosstalk between ROS and plant hormones in seed germination., (© 2014 John Wiley & Sons Ltd.)

Orthodox seeds are living organisms that survive anhydrobiosis and may display dormancy, an inability to germinate at harvest. Seed germination potential can be acquired during a prolonged period of dry storage called after-ripening. The aim of this work was to determine if gene transcription is an underlying regulatory mechanism for dormancy alleviation during after-ripening. To identify changes in gene transcription strictly associated with the acquisition of germination potential but not with storage, we used seed storage at low relative humidity that maintains dormancy as control. Transcriptome profiling was performed using DNA microarray to compare change in gene transcript abundance between dormant (D), after-ripened non-dormant (ND) and after-ripened dormant seeds (control, C). Quantitative real-time polymerase chain reaction (qPCR) was used to confirm gene expression. Comparison between D and ND showed the differential expression of 115 probesets at cut-off values of two-fold change (p<0.05). Comparisons between both D and C with ND in transcript abundance showed that only 13 transcripts, among 115, could be specific to dormancy alleviation. qPCR confirms the expression pattern of these transcripts but without significant variation between conditions. Here we show that sunflower seed dormancy alleviation in the dry state is not related to regulated changes in gene expression.

Sunflower (Helianthus annuus L.) seed viability is affected by moisture content (MC) during ageing and is related to accumulation of hydrogen peroxide and changes in energy metabolism. The aim of the present work was to investigate the effect of ageing on DNA alteration events by RAPD (random amplification of polymorphic DNA) analysis and to determine whether loss of seed viability might correspond to a controlled programmed cell death (PCD). Ageing of sunflower seeds was carried out at 35 °C for 7 d at different MCs. The higher the MC, the lower was the seed viability. RAPD analysis showed that DNA alterations occurred during ageing especially in seeds containing a high MC. In addition, PCD, as revealed by DNA fragmentation and TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick-end labelling) assay, was detected in aged seeds at MCs which resulted in ∼50% seed viability. At the cellular level, TUNEL assay and propidium iodide staining showed that cell death concerns all the cells of the embryonic axis. The quantification of the adenylate pool highlights mitochondrial dysfunction in aged seeds containing a high MC. The involvement of oxidative burst, mitochondria dysfunction, and PCD in seed loss of viability is proposed.

Ageing induces seed deterioration expressed as the loss of seed vigour and/or viability. Priming treatment, which consists in soaking of seeds in a solution of low water potential, has been shown to reinvigorate aged seeds. We investigate the importance of catalase in oxidation protection during accelerated ageing and repair during subsequent priming treatment of sunflower (Helianthus annuus L.) seeds. Seeds equilibrated to 0.29g H2Og(-1) dry matter (DM) were aged at 35°C for different durations and then primed by incubation for 7 days at 15°C in a solution of polyethylene glycol 8000 at -2MPa. Accelerated ageing affected seed germination and priming treatment reversed partially the ageing effect. The inhibition of catalase by the addition of aminotriazol during priming treatment reduced seed repair indicating that catalase plays a key role in protection and repair systems during ageing. Ageing was associated with H2O2 accumulation as showed by biochemical quantification and CeCl3 staining. Catalase was reduced at the level of gene expression, protein content and affinity. Interestingly, priming induced catalase synthesis by activating expression and translation of the enzyme. Immunocytolocalization of catalase showed that the enzyme co-localized with H2O2 in the cytosol. These results clearly indicate that priming induce the synthesis of catalase which is involved in seed recovery during priming., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

After-ripening is the mechanism by which dormant seeds become nondormant during their dry storage after harvest. The absence of free water in mature seeds does not allow detectable metabolism; thus, the processes associated with dormancy release under these conditions are largely unknown. We show here that sunflower (Helianthus annuus) seed alleviation of dormancy during after-ripening is associated with mRNA oxidation and that this oxidation is prevented when seeds are maintained dormant. In vitro approaches demonstrate that mRNA oxidation results in artifacts in cDNA-amplified fragment length polymorphim analysis and alters protein translation. The oxidation of transcripts is not random but selective, and, using microarrays, we identified 24 stored mRNAs that became highly oxidized during after-ripening. Oxidized transcripts mainly correspond to genes involved in responses to stress and in cell signaling. Among them, protein phosphatase 2C PPH1, mitogen-activated protein kinase phosphatase 1, and phenyl ammonia lyase 1 were identified. We propose that targeted mRNA oxidation during dry after-ripening of dormant seeds could be a process that governs cell signaling toward germination in the early steps of seed imbibition.

The physiological dormancy of sunflower (Helianthus annuus) embryos can be overcome during dry storage (after-ripening) or by applying exogenous ethylene or hydrogen cyanide (HCN) during imbibition. The aim of this work was to provide a comprehensive model, based on oxidative signaling by reactive oxygen species (ROS), for explaining the cellular mode of action of HCN in dormancy alleviation. Beneficial HCN effect on germination of dormant embryos is associated with a marked increase in hydrogen peroxide and superoxide anion generation in the embryonic axes. It is mimicked by the ROS-generating compounds methylviologen and menadione but suppressed by ROS scavengers. This increase results from an inhibition of catalase and superoxide dismutase activities and also involves activation of NADPH oxidase. However, it is not related to lipid reserve degradation or gluconeogenesis and not associated with marked changes in the cellular redox status controlled by the glutathione/glutathione disulfide couple. The expression of genes related to ROS production (NADPHox, POX, AO1, and AO2) and signaling (MAPK6, Ser/ThrPK, CaM, and PTP) is differentially affected by dormancy alleviation either during after-ripening or by HCN treatment, and the effect of cyanide on gene expression is likely to be mediated by ROS. It is also demonstrated that HCN and ROS both activate similarly ERF1, a component of the ethylene signaling pathway. We propose that ROS play a key role in the control of sunflower seed germination and are second messengers of cyanide in seed dormancy release.

Reactive Oxygen Species (ROS) are continuously produced during seed development, from embryogenesis to germination, but also during seed storage. ROS play a dual role in seed physiology behaving, on the one hand, as actors of cellular signaling pathways and, on the other hand, as toxic products that accumulate under stress conditions. ROS, provided that their amount is tightly regulated by the balance between production and scavenging, appear now as being beneficial for germination, and in particular to act as a positive signal for seed dormancy release. Such an effect might result from the interplay between ROS and hormone signaling pathways thus leading to changes in gene expression or in cellular redox status. We also propose that changes in ROS homeostasis would play a role in perception of environmental factors by seeds during their germination, and thus act as a signal controlling the completion of germination. However, uncontrolled accumulation of ROS is likely to occur during seed aging or seed desiccation thus leading to oxidative damage toward a wide range of biomolecules and ultimately to necroses and cell death. We present here the concept of the "oxidative window for germination", which restricts the occurrence of the cellular events associated with germination to a critical range of ROS level, enclosed by lower and higher limits. Above or below the "oxidative window for germination", weak or high amounts of ROS, respectively, would not permit progress toward germination.

Freshly harvested sunflower (Helianthus annuus L.) seeds are considered to be dormant because they fail to germinate at relatively low temperatures (10 degrees C). This dormancy results mainly from an embryo dormancy and disappears during dry storage. Although endogenous ethylene is known to be involved in sunflower seed alleviation of dormancy, little attention had been paid to the possible role of cyanide, which is produced by the conversion of 1-aminocyclopropane 1-carboxylic acid to ethylene, in this process. The aims of this work were to investigate whether exogenous cyanide could improve the germination of dormant sunflower seeds and to elucidate its putative mechanisms of action. Naked dormant seeds became able to germinate at 10 degrees C when they were incubated in the presence of 1 mM gaseous cyanide. Other respiratory inhibitors showed that this effect did not result from an activation of the pentose phosphate pathway or the cyanide-insensitive pathway. Cyanide stimulated germination of dormant seeds in the presence of inhibitors of ethylene biosynthesis, but its improving effect required functional ethylene receptors. It did not significantly affect ethylene production and the expression of genes involved in ethylene biosynthesis or in the first steps of ethylene signalling pathway. However, the expression of the transcription factor Ethylene Response Factor 1 (ERF1) was markedly stimulated in the presence of gaseous cyanide. It is proposed that the mode of action of cyanide in sunflower seed dormancy alleviation does not involve ethylene production and that ERF1 is a common component of the ethylene and cyanide signalling pathways.

Recent advances in plant physiology signaling pathways have led to consider reactive oxygen species (ROS) as being key actors in the regulation of germination and dormancy. ROS accumulation during seed dry storage or during their imbibition would trigger cellular events controlling the realization of germination. We show that ROS accumulation triggers specific carbonylation of proteins thus modifying the occurrence of enzyme-mediated reactions during germination or facilitating reserve protein degradation through the proteasome. This suggests that dormancy is in part controlled by protein oxidation. ROS can also act as a positive signal in seed dormancy release through their effect on other mechanisms such as the control of the cellular redox status and the activation of transcription factors. Their interaction with abscisic acid and gibberellins is also evoked and a new mechanism of dormancy regulation in which ROS crosstalk with hormonal pathways is proposed.

At harvest, sunflower (Helianthus annuus L.) seeds are dormant and unable to germinate at temperatures below 15 degrees C. Seed storage in the dry state, known as after-ripening, is associated with an alleviation of embryonic dormancy allowing subsequent germination at suboptimal temperatures. To identify the process by which dormancy is broken during after-ripening, we focused on the role of reactive oxygen species (ROS) in this phenomenon. After-ripening entailed a progressive accumulation of ROS, namely superoxide anions and hydrogen peroxide, in cells of embryonic axes. This accumulation, which was investigated at the cellular level by electron microscopy, occurred concomitantly with lipid peroxidation and oxidation (carbonylation) of specific embryo proteins. Incubation of dormant seeds for 3 h in the presence of hydrogen cyanide (a compound that breaks dormancy) or methylviologen (a ROS-generating compound) also released dormancy and caused the oxidation of a specific set of embryo proteins. From these observations, we propose a novel mechanism for seed dormancy alleviation. This mechanism involves ROS production and targeted changes in protein carbonylation patterns.